JP3146778B2 - Buffer solution and method for measuring alkaline phosphatase activity using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a compound of the general formula

[0002]

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An amine derivative represented by the formula (wherein R ¹ represents hydrogen or a substituted or unsubstituted alkyl group;
The alkyl group has 1 to 4 carbon atoms, and R ² and R ³ are
Represents a substituted or unsubstituted alkyl group, and the alkyl group has 1 to 4 carbon atoms);

[0004]

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[0005] An aminosulfonic acid derivative represented by the formula (wherein, R ⁵ and R ⁶ represent hydrogen or a substituted or unsubstituted alkyl group, the alkyl group has 1 to 4 carbon atoms, and R ⁷ is a sulfo group. Represents a substituted alkyl group,
Wherein the alkyl group has 1 to 4 carbon atoms) and a method for measuring the activity of alkaline phosphatase using the same.

[0006]

2. Description of the Related Art Conventionally, buffers used for measuring alkaline phosphatase activity contained in living organisms include sodium carbonate-sodium hydrogen carbonate, diethanolamine-hydrochloric acid, 2-amino-2-methylpropanol-hydrochloric acid. , 2-ethylaminoethanol-hydrochloric acid and the like, all of which were used at a high concentration of 0.5M to 1.5M.

[0007]

However, when measuring an extremely small amount of alkaline phosphatase activity, the alkaline phosphatase activity decreases with the above-mentioned high-concentration buffer, resulting in a decrease in measurement sensitivity. For this reason, a method of measuring by lowering the concentration of the buffer solution has been adopted. However, when the concentration of the buffer solution is reduced, the buffer capacity decreases, and the pH deviates from the set condition due to the influence of carbon dioxide gas during storage. Therefore, at present, a decrease in alkaline phosphatase activity over time is observed. Then, the appearance of a buffer solution free from such problems has been expected.

[0008]

Means for Solving the Problems The inventors of the present invention have worked diligently to find a buffer solution which can obtain a highly sensitive and stable measurement result, and as a result, have found a buffer solution which is capable of performing a stable and highly sensitive measurement. Is completed.

The present invention provides a compound represented by the general formula

[0010]

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An amine derivative represented by the formula (wherein R ¹ represents hydrogen or a substituted or unsubstituted alkyl group;
The alkyl group has 1 to 4 carbon atoms, and R ² and R ³ are
Represents a substituted or unsubstituted alkyl group, and the alkyl group has 1 to 4 carbon atoms);

[0012]

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An aminosulfonic acid derivative represented by the formula (wherein R ⁵ and R ⁶ represent hydrogen or a substituted or unsubstituted alkyl group, the alkyl group has 1 to 4 carbon atoms, and R ⁷ is a sulfo group Represents a substituted alkyl group,
Wherein the alkyl group has 1 to 4 carbon atoms) and a method for measuring the activity of alkaline phosphatase using the same.

Examples of the amine derivative represented by the general formula (I) constituting the buffer solution of the present invention include diethanolamine (hereinafter abbreviated as DEA), dimethylamine (hereinafter abbreviated as DMA), and diethylamine (hereinafter abbreviated as DEtA).
), 2-ethylaminoethanol (hereinafter abbreviated as EAE), triethylamine (hereinafter abbreviated as TEtA), triethanolamine (hereinafter abbreviated as TEA), N-ethyldiethanolamine (hereinafter abbreviated as EDEA), diisopropylamine (hereinafter abbreviated as DIPA) Abbreviated).

The aminosulfonic acid derivative represented by the general formula (II) includes N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (hereinafter referred to as B)
ES), N-cyclohexyl-3-aminopropanesulfonic acid (hereinafter abbreviated as CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (hereinafter abbreviated as CAPSO), N-cyclohexyl-2
-Aminoethanesulfonic acid (hereinafter abbreviated as CHES), 3
-{N, N-bis (2-hydroxyethyl) amino}
-2-Hydroxypropanesulfonic acid (hereinafter DIPS)
O), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter abbreviated as TAPS), 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter T)
APSO), N-tris (hydroxymethyl)
Methyl-2-aminoethanesulfonic acid (hereinafter abbreviated as TES) and the like can be mentioned.

The aminosulfonic acid derivatives represented by the general formula (III) include 3- {4- (2-hydroxyethyl) -1-piperazinyl} propanesulfonic acid (hereinafter abbreviated as EPPS), 2- { 4- (2-hydroxyethyl) -1-piperazinyl} ethanesulfonic acid (hereinafter abbreviated as HEPES), 2-hydroxy-3-
{4- (2-hydroxyethyl) -1-piperazinyl} propanesulfonic acid (hereinafter abbreviated as HEPPSO),
Piperazine-1,4-bis (2-ethanesulfonic acid)
(Hereinafter abbreviated as PIPES), piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (hereinafter abbreviated as POPSO), and the like.

The aminosulfonic acid derivatives represented by the general formula (IV) include 2-morpholinoethanesulfonic acid (hereinafter abbreviated as MES), 3-morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS), 2-hydroxy- 3-morpholinopropanesulfonic acid (hereinafter MOPSO
Abbreviated).

In the present invention, the enzyme activity of alkaline phosphatase is measured using the above-mentioned buffer. The method for measuring enzyme activity in the present invention is described in, for example, Clin. Che
m. 23/12, 2236-2274 (1977). At this time, the buffer described above can be used as a substrate solution.

Substrates which can be used in carrying out the above-mentioned measuring method are represented by the general formula

[0020]

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Wherein X represents H, Na, K or NH ₄ , and Y represents H, Cl, Br, a methyl group or a methoxy group.

As the 1,2-dioxetane derivative represented by the general formula (V), for example,

[0023]

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(Where X is the same as above) (hereinafter abbreviated as AMPPD),

[0025]

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(Where X is the same as above) (hereinafter abbreviated as CSPD),

[0027]

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(Wherein X is the same as described above).

In carrying out the method, a general formula

[0030]

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An organic polymer represented by the formula (wherein n is 30
６００600, R ⁸ , R ⁹ and R ¹⁰ represent a phenyl group or a substituted or unsubstituted alkyl group, the alkyl group has 1 to 18 carbon atoms, and Z represents Cl, Br or I. ) Is preferably carried out in the presence of a sensitizer represented by As the sensitizer represented by the general formula (VI), for example, R ⁸ , R ⁹ and R ¹⁰ are butyl groups and n is 30
Organic polymer (hereinafter referred to as TB)
Q), an organic polymer in which R ⁹ is a benzyl group, R ⁸ and R ¹⁰ are methyl groups, n is 30 to 600, and Z is Cl (hereinafter abbreviated as BDMQ).

[0032]

【Example】

Example _{-1 1.0mM-MgCl 2, 0.05%} -NaN 3, 0.
PH containing 02% -AMPPD, 0.04% -BDMQ
A buffer solution of = 10.00 ± 0.05 was prepared as shown in Table 1.

[0033]

[Table 1]

Example 2 The substrate buffer No. 1 prepared in Example 1 was used. 1 to No. 9,
Add 200 μl of each substrate buffer to alkaline phosphatase (1
ng / ml), and allowed to stand at room temperature for 20 minutes. Then, the alkaline phosphatase activity was measured using Clinyl-Mat. The results are shown in Table-2.

[0035]

[Table 2]

Example 3 1.0 mM MgCl ₂ , 0.05% NaN ₃ , 0.1 mM
Various pH and 0.1M DEA-BES buffers containing 02% -AMPPD and 0.04% -BDMQ were prepared as shown in Table-3.

[0037]

[Table 3]

Example 4 Immunoassay for α-fetoprotein was carried out using anti-human α-fetoprotein-immobilized magnetic particles and an anti-human α-fetoprotein alkaline phosphatase label, and the enzyme reaction was prepared in Example-3. Alkaline phosphatase activity was measured using the six types of substrate buffers thus obtained. The result is shown in FIG.

Comparative Example 1 1.0 mM MgCl ₂ , 0.05% NaN ₃ , 0.1 mM
Various pH and 0.1M DEA-HCl buffers containing 02% -AMPPD and 0.04% -BDMQ were prepared as shown in Table-4.

[0040]

[Table 4]

Comparative Example 2 Immunoassay for α-fetoprotein was performed using anti-human α-fetoprotein-immobilized magnetic particles and labeled anti-human α-fetoprotein alkaline phosphatase, and the enzyme reaction was prepared in Comparative Example-1. Alkaline phosphatase activity was measured using the six types of substrate buffers thus obtained. The result is shown in FIG.

Example-5 Substrate buffer No. prepared in Example-1 3 and No. 3
4 was prepared as in Table-5.

[0043]

[Table 5]

Example -6 An immunoassay for α-fetoprotein was carried out using anti-human α-fetoprotein-immobilized magnetic particles and an anti-human α-fetoprotein alkaline phosphatase label, and the enzyme reaction was prepared in Example-5. Alkaline phosphatase activity was measured using the six types of substrate buffers thus obtained. The result is shown in FIG.

Comparative Example 3 Substrate buffer No. prepared in Example 1 1 and No.
2 was prepared as in Table-6.

[0046]

[Table 6]

Comparative Example 4 Immunoassay for α-fetoprotein was performed using anti-human α-fetoprotein-immobilized magnetic particles and labeled anti-human α-fetoprotein alkaline phosphatase, and the enzyme reaction was prepared in Comparative Example-3. Alkaline phosphatase activity was measured using the six types of substrate buffers thus obtained. The result is shown in FIG.

COMPARATIVE EXAMPLE-5 Substrate buffer No. prepared in Example-1 1 at 37 ° C
Weekly loading was performed.

Comparative Example-6 An immunoassay for TSH was carried out using anti-human TSH-immobilized magnetic particles and an anti-human TSH alkaline phosphatase label, and the enzymatic reaction was carried out in Comparative Example-5. Was used to measure alkaline phosphatase activity. As a control, the same operation as above was performed using an unloaded substrate buffer. The results are shown in FIG.

Example -7 Substrate buffer No. prepared in Example 1 9 at 37 ° C for 1
Weekly loading was performed.

Example -8 An immunoassay for TSH was carried out using anti-human TSH-immobilized magnetic particles and an anti-human TSH alkaline phosphatase label, and the enzymatic reaction was carried out in Example -7. Was used to measure alkaline phosphatase activity. As a control, the same operation as above was performed using an unloaded substrate buffer. FIG. 6 shows the result.

Comparative Example-7 The substrate buffer No. 1 prepared in Example-1 was used. 1 was left open at 4 ° C. for 3 days and 7 days with the lid open, a carbon dioxide gas absorption load was performed, and the pH was measured. The result is shown in FIG.

Example -9 The substrate buffer No. prepared in Example 1 was used. 9 was left at 4 ° C. for 3 days and 7 days with the lid open, a carbon dioxide gas absorption load was performed, and the pH was measured. The result is shown in FIG.

[0054]

According to the method of the present invention, the compounds represented by the above general formulas (I), (II), (III) and
By using the substrate buffer represented by (IV), highly sensitive and stable alkaline phosphatase activity can be measured. This can be performed with a sensitivity 1.4 to 1.6 times higher than the conventional method. In addition, p
The change in H is as small as 5/9.

[Brief description of the drawings]

FIG. 1 is a diagram showing the measurement of optimal pH performed in Example-4.

FIG. 2 is a diagram showing an optimum pH measured in Comparative Example-2.

FIG. 3 is a diagram illustrating a measurement of a buffer solution concentration performed in Example-6.

FIG. 4 is a diagram illustrating the measurement of buffer concentration performed in Comparative Example-4.

FIG. 5 is a diagram illustrating the measurement of substrate stability under a temperature load performed in Comparative Example-6.

FIG. 6 is a diagram illustrating the measurement of substrate stability under a temperature load performed in Example-8.

FIG. 7 is a diagram showing a measurement of a change in pH due to carbon dioxide in the air performed in Comparative Example-7.

FIG. 8 is a diagram illustrating a measurement of a change in pH due to carbon dioxide in air performed in Example-9.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) C12Q 1/00-1/66

Claims (7)

(57) [Claims] 1. A compound of the general formula Wherein R ¹ represents hydrogen or a substituted or unsubstituted alkyl group, the alkyl group has 1 to 4 carbon atoms, and R ² and R ³ represent a substituted or unsubstituted alkyl group And the alkyl group has 1 carbon atom
To 4), and a general formula: Wherein R ⁵ and R ⁶ represent hydrogen or a substituted or unsubstituted alkyl group, the alkyl group has 1 to 4 carbon atoms, and R ⁷ is substituted with a sulfo group. An alkyl group, wherein the alkyl group has 1 to 4 carbon atoms). 2. A compound of the general formula Wherein X is H, Na, K or NH
_{4 wherein} Y represents H, Cl, Br, a methyl group or a methoxy group). 3. The method according to claim 1, wherein the substrate is The method according to claim 2, wherein X is the same as described above. 4. The method according to claim 1, wherein the substrate is The method according to claim 2, wherein X is the same as described above. 5. A sensitizer represented by the following general formula: Wherein n is 30 to 600, R ⁸ , R ⁹ and R ¹⁰ represent a benzyl group or a substituted or unsubstituted alkyl group, and the alkyl group has 1 to 18 carbon atoms. And Z represents Cl, Br or I.). (6) a sensitizer comprising: The method according to claim 2, wherein n is 30 to 600 and Z is Cl. 7. The sensitizer according to claim 1, wherein: The method according to claim 2, wherein n is 30 to 600 and Z is Cl.